Method for promoting electrogenesis of electrochemical active bacteria by utilizing electrodeposited chitosan

Abstract

A nanometer material modified electrode is an effective means for improving adhesion of electrochemical active bacteria and generating electricity, chitosan is often used as basic modification, but the influence of the chitosan on the electrochemical active bacteria is not clear all the time. The invention discloses a method for promoting electrogenesis of electrochemical active bacteria by utilizing electrodeposited chitosan. The promotion effect of chitosan modification on electrogenesis of the electrochemical active bacteria is not neglected, and the electrogenesis of the electrochemical active bacteria can be improved by 14.19 times at most. The promotion effect of the electrodeposition time of the chitosan on electricity generation of the electrochemical active bacteria is in an inverted U shape, and the promotion effect is optimal when the electrodeposition time is 10 min. The improvement of the electricity generation of the electrochemical active bacteria by the chitosan sediment comes from slight improvement of the electrochemical activity and obvious increase of the biomass. When the electrodeposition time is short, the amino content of chitosan sediment is insufficient, and adhesion of electrochemical active bacteria cannot be effectively promoted. On the contrary, when the electrodeposition time is relatively long, the conversion from the amino group to the pyridine nitrogen begins to appear, and the promotion effect of the amino group on the attachment of the electrochemical active bacteria is weakened.

Description

Technical Field

[0001] This invention belongs to the field of microbial electrochemistry technology, and particularly relates to a method for promoting electrochemically active bacteria to generate electricity through in-situ modified electrodes. Background Technology

[0002] Electrochemically active bacteria are a class of environmental microorganisms with unique energy metabolism pathways and extracellular respiration capabilities. Their bioelectricity generation capacity offers broad prospects for developing compact, sustainable power supply systems, such as powering remote sensors or wearable devices. Furthermore, since the generation of bioelectric signals by electrochemically active bacteria is related to cellular metabolism, a series of biosensors based on electrochemically active bacteria have been developed for detecting water quality indicators (such as biotoxicity and BOD). Generally, biosensors based on electrochemically active bacteria typically utilize mature biofilms. This is because mature biofilms of electrochemically active bacteria exhibit high electrical signal strength, but the cultivation process requires several days or even weeks, limiting applications in scenarios such as real-time detection. Although early-stage biofilm preparation by electrochemically active bacteria is faster, its electrical signal is relatively weak. Therefore, how to rapidly obtain electrochemically active bacterial biofilms with high power generation remains a pressing problem to be solved.

[0003] To address this issue, previous studies have reported various electrode modification techniques aimed at enhancing the electrogenic capacity of electrochemically active bacteria. The most common approach involves modifying electrodes with nanomaterials (such as carbon nanotubes and graphene), which increases specific surface area and biofilm biomass. Simultaneously, conductive polymer modification of electrode surfaces has also been widely applied. Conductive polymers not only increase specific surface area and improve hydrophilicity, but their unique morphology also enhances cell adhesion, while their nanowire structures improve electron transfer efficiency between cells and electrodes. Recently, metal oxide modification has emerged as a novel, low-cost strategy, acting as both a redox medium and accelerating heterogeneous electron transfer. Notably, these common electrode modifications often use chitosan (CTS) as the base modifier because it prevents nanomaterial aggregation and enhances biocompatibility. However, since CTS is often used synergistically with other modifiers, its impact on the adhesion of electrochemically active bacteria is often overlooked, and its mechanism of action remains unclear.

[0004] In fact, the effects of CTS on electrochemically active bacteria are likely complex. First, CTS contains an amino group, which protonates in acidic environments (pH < 6.5). Amino protonation is expected to increase the positive charge on the electrode surface, enhance the electrode's positive charge, promote the attachment of electrochemically active bacteria, and increase biomass. However, amino protonation is also the basis of CTS's antibacterial activity. For example, a concentration of 0.50 mg / mL of CTS can inhibit the growth of *E. coli*. Its antibacterial mechanism lies in the fact that CTS binds tightly to the bacterial cell wall through electrostatic interactions, leading to disruption of the cell membrane lipid layer. Furthermore, most electrochemically active bacteria are Gram-negative, and CTS exhibits higher binding selectivity for Gram-negative bacteria. Second, CTS can improve the smoothness of the electrode surface, enhancing the contact between the electrochemically active bacteria and the electrode. However, CTS has poor conductivity, and excessive deposition on the electrode surface may hinder heterogeneous electron transfer between the electrochemically active bacteria and the electrode. Finally, CTS deposits may occupy the pores between carbon fibers, leading to narrowing of mass transfer channels and reduced substrate transport efficiency. Therefore, systematic research is urgently needed to elucidate the effects of CTS on electrochemically active bacteria's electricity generation and its mechanism of action. Summary of the Invention

[0005] To address this issue, this patent discloses the effect and biological mechanism of electrodeposited CTS on electrochemically active bacteria's electricity generation, and reports a method for promoting electricity generation by electrodeposited CTS, as follows:

[0006] 1. A method for promoting electrochemically active bacteria to generate electricity using electrodeposition of CTS. The principle of this method is to deposit CTS on the electrode surface using electrodeposition. The amino groups contained in the CTS deposit can increase the positive charge of the electrode after protonation, promote the attachment of electrochemically active bacteria, increase the biomass of electrochemically active bacteria on the electrode surface, and improve the electricity generation of electrochemically active bacteria.

[0007] 2. The optimized electrodeposition time is 10 min. Electrodeposition time less than 10 min will result in a low amino content on the electrode surface, while electrodeposition time more than 10 min will lead to the conversion of amino groups on the electrode surface into pyridine nitrogen and pyrrole nitrogen, weakening the promoting effect of electrodeposited CTS on electrochemically active bacteria electricity generation.

[0008] 3. The solution used for electrodeposition of CTS is 1 g / L CTS solution. CTS is electrodeposited using the chronoamperometry method. The electrodeposition is performed using the chronoamperometry method, and the potential is set to -1.5V, with reference to the Ag / AgCl electrode.

[0009] 4. The electrochemically active bacteria used were the model strain Shewanella loihica PV-4. Attached Figure Description

[0010] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0011] Figure 1 This section describes the changes in electrode surface characteristics during the CTS electrodeposition process in Example 1. It includes in-situ FTIR analysis of CTS electrodeposition at different time points (a), X-ray photoelectron spectroscopy (XPS) elemental analysis (b), scanning electron microscope (SEM) images of blank carbon cloth (cd), SEM images of CTS-modified carbon cloth after 60 min (ef), and energy-dispersive X-ray spectroscopy (EDS) images of N and O elements (g, h).

[0012] Figure 2 This example illustrates the effect of carbon cloth surface electrodeposition CTS on the power generation performance of S. loihica PV-4 in Example 1. The results include the t curve (a), CV curve (b), first derivative of the CV curve (c), limiting current of the CV curve (d), EIS spectrum (e), and charge transfer resistance (f).

[0013] Figure 3 SEM images of the electrode surface under different electrodeposition times in Example 2 are shown. These include unmodified electrode (a), electrodeposition for 1 min (b), electrodeposition for 5 min (c), electrodeposition for 10 min (d), electrodeposition for 30 min (e), and electrodeposition for 60 min (f).

[0014] Figure 4 This section describes the effects of different CTS electrodeposition times on the power generation and biomass of S. loihica PV-4 in Example 2. The results include the t curve (a), the CV curve using potassium ferricyanide / potassium ferrocyanide as the redox medium (b), the limiting current of the CV curve (c), and the protein content of S. loihica PV-4 cells (d).

[0015] Figure 5 Metabolic activity and energy metabolism levels of *S. loihica* PV-4 bacteria in Example 2. This includes confocal laser scanning microscopy (CLSM) images of *S. loihica* PV-4 attached to the electrode at the time of CTS electrodeposition (a), 1 min (b), 10 min (c), and 60 min (d); average fluorescence intensity of the CLSM images (e); single-cell ATP content of *S. loihica* PV-4 (f); single-cell electron transport system activity (ETSA) level of *S. loihica* PV-4 (g); and single-cell NADH content (h).

[0016] Figure 6This illustrates the effect of different CTS electrodeposition times on electrode surface properties in Example 3. This includes porosity (a), Zeta potential (b), and the contact angle of the carbon cloth after unmodified electrode (c), 1 min CTS electrodeposition (d), 5 min CTS electrodeposition (e), 10 min CTS electrodeposition (f), 30 min CTS electrodeposition (g), and 60 min CTS electrodeposition (h).

[0017] Figure 7 The changes in functional groups on the electrode surface after different CTS electrodeposition times in Example 3 are shown. This includes the functional group ratio (a) and the true and fitted XPS spectra of N and O on the CTS electrodeposited electrodes at different times (b), 1 min (c), 5 min (d), 10 min (e), 30 min (f), and 60 min (g). Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels;

[0021] Among them, the ATCC number of Shewanella loihica PV-4 strain is BAA-1088.

[0022] The multiple linear regression analysis of electrode surface functional groups and electrochemically active bacterial biomass in Example 3 of this invention is shown in Table 1:

[0023] Table 1

[0024]

[0025]

[0026] Example 1

[0027] The promoting effect of electrodeposited CTS on electrochemically active bacteria's electricity generation includes the following steps:

[0028] (1) First, an electrochemical cell (EC) with identical structure was constructed to ensure the reproducibility of electrode preparation. The EC contained a working electrode (WE), a counter electrode (CE), and a reference electrode (RE). The working electrode was a 2cm*2cm piece of treated carbon cloth, the counter electrode was a 2cm*2cm*1mm Pt sheet, and the reference electrode was an Ag / AgCl electrode (0.2224V relative to the standard hydrogen electrode). The entire electrochemical cell system was 50mL in size.

[0029] (2) Prepare the electrolyte for electrodeposition of CTS by dissolving 1g of CTS in 1L of acetic acid (2%) to obtain a 1g / L CTS solution.

[0030] (3) CTS electrodeposition was performed in an electrochemical cell using the chronoamperometry method, with a potential set at -1.5 V and a time set at 60 min. The RE electrode was used as the reference electrode for all potentials. The working electrodes before and after CTS electrodeposition were characterized by FTIR, XPS, SEM, and EDS, respectively. The results are as follows: Figure 1 As shown.

[0031] (4) The effect of electrodeposited CTS on electrochemically active bacteria power generation was analyzed using the electrochemically active bacterial strain Shewanella loihica PV-4. First, S. loihica PV-4 was revived and cultured in LB medium at 22℃ and 150 rpm for 36 h. After S. loihica PV-4 entered the plateau phase, the bacterial suspension was centrifuged at 5000 rpm and 22℃ for 15 min. After centrifugation, the precipitate was resuspended in aerated DM medium to obtain a DM bacterial suspension (OD = 1). Each liter of LB medium contained 10 g peptone, 10 g NaCl, and 5 g yeast extract. Each liter of DM solution contained 2.50 g sodium bicarbonate, 0.07 g anhydrous calcium chloride, 1.00 g ammonium chloride, 0.09 g anhydrous magnesium chloride, 10.00 g sodium chloride, and 7.20 g HEPES (4-hydroxyethylpiperazine ethanesulfonic acid).

[0032] (5) Early biofilm of S. loihica PV-4 was prepared by filtration. 100 mL of S. loihica PV-4 bacterial suspension was passed through unmodified carbon cloth or electrodeposited CTS modified carbon cloth to prepare biofilm.

[0033] (6) After preparation, a microbial electrochemical cell (MEC) was constructed using carbon cloth as the working electrode. The cell body, reference electrode, and working electrode of the MEC were the same as those of the aforementioned system. The electrogenic performance of *S. loihica* PV-4 was evaluated by running the MEC: electrochemical activity was analyzed using chronoamperometry, with the working electrode potential set at 0.5V; the onset potential and limiting current of electrochemically active bacteria were determined using cyclic voltammetry. The potential scan range was -0.4V to 0.6V, the scan rate was 5mV / s, and a total of 3 cycles were performed. Electrochemical impedance spectroscopy was used to analyze the electrochemical impedance and composition. The working potential was set at 0.5V, the voltage amplitude was 5mV, and the frequency range was 50mHz to 100kHz. A typical equivalent circuit model R was used. s +R ct / Q is used to fit the EIS spectrum, where R s R ct Q and Q represent ohmic resistance, charge transfer resistance, and double-layer capacitance, respectively. The effects of 60 min electrodeposition of CTS on the charge production of electrochemically active bacteria were analyzed, and the results are as follows: Figure 2 As shown.

[0034] like Figure 1 As shown in a, two distinct infrared peaks appear after CTS electrodeposition, with peak values ​​located at 1090 and 1550 cm⁻¹, respectively. -1 The two peaks, CO bonds and protonated amino groups, respectively, represent the CO bonds and protonated amino groups present in CTS, indicating that CTS was deposited in situ on the carbon cloth surface. XPS analysis showed that after 60 min of CTS electrodeposition, the nitrogen and oxygen content in the sample increased from 2.45±0.04% and 7.08±0.40% to 8.14±0.66% and 25.34±0.18%, respectively. Figure 1 b). EDS analysis also showed enrichment of nitrogen and oxygen in the sediments. Figure 1 (g and h). These results further confirm the phenomenon of CTS deposits forming on the surface of carbon fiber cloth. Notably, the deposits after 60 min of CTS electrodeposition exhibited irregular morphology, with some deposits adhering to the carbon fibers and occupying the pores of the carbon cloth, indicating that the electrodeposition time may have been too long.

[0035] like Figure 2As shown in figure a, the early biofilm of *S. loihica* PV-4 attached to the unmodified carbon cloth generated a weak current of 2.21 ± 0.23 μA. After CTS electrodeposition, the current value of the early biofilm of *S. loihica* PV-4 increased to 11.02 ± 1.67 μA, an increase of 398.64%. Furthermore, the onset potential of electrochemically active bacteria also changed. The onset potential of the early biofilm on the unmodified electrode was 0.4 V, while it decreased to 0.15 V after CTS electrodeposition, indicating that current generation led to a decrease in overpotential. Figure 2 bc). The limiting current is defined as the current value at a potential of 0.6V, which increased by 41.37% after CTS deposition modification. Figure 2 d). R ct The charge transfer resistance, representing the electricity generated by electrochemically active bacteria, decreased from 11.45±0.49kΩ to 4.46±0.01kΩ. Figure 2 However, SEM observation showed that excessive CTS buildup was observed on the electrode surface after 60 minutes of electrodeposition, indicating that the CTS electrodeposition time needs further optimization. Figure 1 cf).

[0036] Example 2

[0037] The effect of CTS electrodeposition time on electrochemically active bacteria's electricity generation includes the following steps:

[0038] (1) CTS was electrodeposited in the same manner as in Example 1. To optimize the formation of CTS deposits, 1 min, 5 min, 10 min, 30 min and 60 min were selected as electrodeposition times.

[0039] (2) To elucidate the mechanism by which CTS electrodeposition time exhibits an inverted U-shaped effect on the electrochemical current generated by electrochemically active bacteria, the electrochemical activity of the electrodes, the total biomass of electrochemically active bacteria, and the energy metabolism level of single cells were analyzed. The results are as follows: Figure 4 As shown.

[0040] (3) Electrochemical activity of the electrodes was determined by cyclic voltammetry (CV), using potassium ferricyanide / potassium ferrocyanide as a redox probe. The total biomass of electrochemically active bacteria was characterized by the protein content on the electrode surface, which was determined using the Bradford method. First, *S. loihica* PV-4 biofilms were obtained by sampling the WE electrode of the MEC. The biofilms were then eluted by shaking at 1500 rpm for 15 minutes. After centrifugation to collect the cell suspension, proteins were extracted using a bacterial protein extraction kit. Finally, the total protein assay kit was used for protein content analysis.

[0041] Such as SEM images ( Figure 3As shown in the figure, the CTS deposit content on the electrode surface decreases significantly with shorter electrodeposition time. When the electrodeposition time is less than 10 min, both excessive accumulation and porosity occupancy disappear. XPS results also confirm that the amount of CTS deposited decreases with shorter electrodeposition time. Figure 1 b) The nitrogen content (representing CTS content) decreased successively to 6.75±0.01% (30 min), 6.60±0.35% (10 min), 5.9±1.09% (5 min), and 5.46±0.03% (1 min). Figure 1 b). Unlike the relationship between CTS content and electrochemically active bacterial electrostatic generation and CTS electrodeposition time, an unexpected trend emerged. Specifically, the current of *S. loihica* PV-4 reached a peak of 22.03 ± 0.57 μA at 10 min electrodeposition time, and the electrochemically active bacterial electrostatic generation showed an inverted U-shaped relationship with CTS electrodeposition time. Figure 4 a) This indicates that specific CTS concentrations can more effectively enhance the electrochemical generation efficiency of electrochemically active bacteria. Notably, after 10 minutes of CTS electrodeposition, the electrochemical generation of active bacteria increased by 14.19 times, a value approaching or even exceeding that of some common nanomaterials. Therefore, these results further demonstrate that the promoting effect of CTS on the electrochemical generation of active bacteria is not negligible. When CTS is used synergistically with nanomaterials, CTS should be used as a positive control to more clearly distinguish the contributions of each component in promoting the electrochemical generation of active bacteria.

[0042] like Figure 4 As shown in b, CTS electrodeposition significantly enhanced the intensity of the potassium ferrocyanide oxidation peak and narrowed the potential difference between the oxidation and reduction peaks. These results indicate that the CTS deposit enhances the electrochemical activity of the electrode and helps reduce the ohmic resistance of electrochemically active bacteria. When the CTS electrodeposition time is 10 min, the electrode electrochemical activity reaches its peak, with an increase of 40.5%. Figure 4 c). However, CTS is far less effective than common nanomaterials such as carbon nanotubes, graphene, and metal oxides in enhancing electrode electrochemical activity. In contrast, CTS electrodeposition has a more significant impact on total biomass—based on total protein, after 10 min of CTS electrodeposition, the biomass of *S. loihica* PV-4 cells on the electrode surface increased by 73.59% (…). Figure 4 d).

[0043] The increased metabolic efficiency of single cells is another reason for the increased current in electrochemically active bacteria. For example... Figure 5As shown, CTS electrodeposition significantly increased ATP content in single cells but did not increase NADH levels. This phenomenon indicates that with CTS deposition, the electron transfer efficiency between electrochemically active bacteria and the electrode, rather than the energy metabolism level of single cells, was improved. The increase in ETSA levels in single cells further confirms this view. Therefore, it can be concluded that electrodeposition of CTS only slightly improves electrode conductivity and electron transfer efficiency of electrochemically active bacteria, but significantly increases biomass, thereby significantly enhancing the electrogenic capacity of electrochemically active bacteria.

[0044] Example 3

[0045] The mechanism by which the inverted U-shaped effect of electrodeposition time on the biomass of electrochemically active bacteria includes the following steps:

[0046] To understand the inverted U-shaped relationship between CTS electrodeposition time and the charge generated by electrochemically active bacteria, material properties were analyzed from four aspects: porosity, conductivity, hydrophilicity, and zeta potential. The results are as follows: Figure 6-7 As shown.

[0047] (1) Porosity was determined by mercury intrusion porosimetry under low and high pressure conditions.

[0048] (2) Electrode conductivity was determined by cyclic voltammetry, using potassium ferricyanide / potassium ferrocyanide as redox probe.

[0049] (3) Hydrophilicity was analyzed by static and dynamic contact angle measurements.

[0050] (4) The zeta potential of S. loihica PV-4 bacterial cells was calculated by electrophoretic light scattering, while the zeta potential of the electrode surface was determined by the flow potential method.

[0051] Electrode porosity and surface hydrophilicity are considered two important factors affecting the biomass on the electrode surface. After 60 min of CTS electrodeposition, the electrode porosity decreased slightly from 78.65 ± 0.34% to 76.75 ± 0.23%. Figure 6 a) This is attributed to the occupancy of inter-carbon fiber pores observed in SEM images. However, the decrease in porosity implies a reduction in biomass, which clearly cannot explain the increase in electrochemically active bacterial biomass on the electrode surface after CTS electrodeposition. Regarding surface hydrophilicity, the unmodified carbon cloth surface exhibits hydrophobicity, while all CTS-modified surfaces become superhydrophilic. Figure 6 The static contact angle reaches 0°. Therefore, the similar superhydrophilic properties exhibited at different CTS electrodeposition times cannot explain the inverted U-shaped effect between electrodeposition time and electrochemically active bacterial biomass. The N element content on the electrode surface represents the CTS content, but the CTS content has an approximately linear relationship with the electrodeposition time (Rch). 2=0.85), which is quite different from the inverted U-shaped relationship between electrodeposition time and electrochemically active bacteria electricity generation.

[0052] Electrodeposition time not only affects CTS content but also alters the type and number of surface functional groups. Specifically, with increasing electrodeposition time, the amino content increased from 2.36±0.10% in the control group to 4.05±0.04% at 1 min, 3.97±0.23% at 5 min, and 5.00±0.30% at 10 min. Figure 7 It is noteworthy that further extending the electrodeposition time could not increase the amino content further, because some nitrogen-containing functional groups were converted to pyridine and pyrrole nitrogen. Protonated amino groups enhance the positive charge of the electrode, while pyridine and pyrrole nitrogen have little effect on electronegativity. Zeta potential analysis further confirmed this difference. Figure 6 As shown in b, the Zeta potentials of S. loihica PV-4 strain and unmodified carbon cloth were -28.70±1.34 mV and -31.27±2.49 mV, respectively, indicating electrostatic repulsion. With increasing amino content, the electrode Zeta potential rose to 0.75±0.23 mV (1 min) and 11.24±0.36 mV (10 min). However, when the electrodeposition time reached 60 min, the Zeta potential dropped to -2.48±0.27 mV, consistent with the decreasing trend of amino content. Therefore, the transfer of amino to pyridine and pyrrole nitrogen is an important reason for the inverted U-shaped effect of biomass.

[0053] Multiple linear regression was used to further explore the association between electrode surface functional groups and biomass. The coefficient of determination of the fitted model reached 0.90 (p = 0.0052). Among these functional groups, the slope of amino groups was 19.36 (p = 0.0187), indicating a significant positive effect on biomass. However, the slopes of pyridine nitrogen and pyrrole nitrogen were -6.51 (p = 0.10) and -8.38 (p = 0.22), respectively, suggesting a possible negative impact on biomass. The slopes of the other two functional groups were relatively low. Therefore, the functional group-based interpretation revealed an inverted U-shaped relationship between CTS electrodeposition time and biomass: CTS electrodeposition increases the amino and zeta potentials on the electrode surface and provides electrostatic attraction to increase biomass; longer CTS electrodeposition induces the conversion of amino groups to pyridine and pyrrole nitrogen, disrupting the biomass-promoting effect of amino groups and leading to a decrease in biomass.

Claims

1. A method for promoting electrochemically active bacteria to generate electricity using electrodeposited chitosan. The principle of this method is to deposit chitosan on the electrode surface using an electrode deposition method. The amino groups contained in the chitosan deposit are protonated, which can increase the positive charge of the electrode, promote the attachment of electrochemically active bacteria, increase the biomass of electrochemically active bacteria on the electrode surface, and improve the electricity generation of electrochemically active bacteria.

2. The method for promoting electrochemically active bacteria to generate electricity using electrodeposited chitosan as described in claim 1, wherein the optimized electrodeposition time is 10 min. An electrodeposition time of less than 10 min will result in a low amino content on the electrode surface, while an electrodeposition time of more than 10 min will result in the conversion of amino groups on the electrode surface to pyridine nitrogen and pyrrole nitrogen, thereby weakening the promoting effect of electrodeposited chitosan on electrochemically active bacteria to generate electricity.

3. The method for promoting electrochemically active bacteria to generate electricity using electrodeposition of chitosan as described in claim 1, wherein the solution used is a 1 g / L chitosan solution, the chitosan is electrodeposited using a chronoamperometry method, the potential is set to -1.5 V, and the potential is referenced to the Ag / AgCl electrode.

4. The method for promoting electrochemically active bacteria to generate electricity using electrodeposited chitosan as described in claim 1, wherein the electrochemically active bacteria is the model strain Shewanella loihica PV-4.

Images